Single-Cell RT-PCR cDNA Subtraction
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A major problem in trying to understand complex developmental processes is one of heterogeneity at both the cellular and molecular levels. At the cellular level it is often difficult to identify cells that are undergoing developmental changes and to establish the stage of differentiation they have reached. At the molecular level, there is then a problem in establishing which of the many thousands of expressed genes are playing a role in development. Several approaches to identifying expressed candidate developmental genes are based on comparing the mRNA expression patterns in cells before and after transition points. Differential screening of cDNA libraries with labeled total cDNA probes from contrasting cell samples (1) provides a simple means of identifying genes that are expressed at high levels in one of the samples. cDNA subtraction protocols (2,3) have increased the sensitivity of this type of approach by removing sequences expressed in both samples. The limitation of these approaches lies in the need for large amounts of starting material necessary for cDNA preparation and subtraction. With the advent of the polymerase chain reaction (PCR) (4) and the development of techniques that allow amplification of target sequences expressed in single cells (5), the limitations of applying cDNA subtraction and differential screening have been removed.
- St. John, T. P. and Davis, R. W. (1979) Isolation of galactose-inducible DNA sequences from Sachromyces cerevisiae by differential plaque filter hybridisation. Cell 16, 443. CrossRef
- Zimmerman, C. R., Orr, W. C., Leclerc, R. F., Barnard, E. C., and Timberlake, W. E. (1980) Molecular cloning and selection of genes regulated in Aspergillus development. Cell 11, 709. CrossRef
- Timberlake, W. E. (1980) Developmental gene regulation in Aspergillus nidulans. Dev. Biol. 78, 497. CrossRef
- Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985) Enzymatic amplification of α-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 239, 1350. CrossRef
- Rappolee, D. A., Wang, A., Mark, D., and Werb, Z. (1989) Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell Biochem. 39, 1–11. CrossRef
- Brady, G., Billia, F., Knox, J., Hoang, T., Kirsch, I. R., Voura, E. B., Hawley, R. G., Cumming, R., Buchwald, M., Siminovitch, K., Miyamoto, N., Boehmelt, G., and Iscove, N. (1995) Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr. Biol. 5, 909–922. CrossRef
- Brady, G., Barbara, M., and Iscove, N. N. (1990) Representative in vitro cDNA amplification from individual hemopoietic cells and colonies. Meth. Mol. Cell. Biol. 2, 17–25.
- Brady, G. and Iscove, N. N. (1993) Amplified representative cDNA libraries from single cells, in Methods in Enzymology, vol. 225 (Wassarman, P. M. and DePamphilis, M. L., eds.), Academic, San Diego, pp. 611–623.
- Trumper, L. H., Brady, G., Bagg, A., Gray, D., Loke, S. L., Griesser, H., Wagman, R., Braziel, R., Gascoyne, R. D., and Vicini, S. (1993) Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood 81, 3097–3115.
- Dulac, C. and Axel, R. (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83, 195–206. CrossRef
- Cumano, A., Paige, C. J., Iscove, N. N., and Brady, G. (1992) Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356, 612–615. CrossRef
- Cano-Gauci, D. F., Lualdi, J. C., Ouellette, A. J., Brady, G., Iscove, N. N., and Buick, R. N. (1993) In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the cryptvillus axis. Exp. Cell Res. 208, 344–349. CrossRef
- Barr, F. G. and Emmanuel, B. S. (1990) Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA. Anal. Biochem. 186, 369–373. CrossRef
- Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
- Single-Cell RT-PCR cDNA Subtraction
- Book Title
- Molecular Embryology
- Book Subtitle
- Methods and Protocols
- Book Part
- pp 601-609
- Print ISBN
- Online ISBN
- Series Title
- Methods in Molecular Biology™
- Series Volume
- Series ISSN
- Humana Press
- Copyright Holder
- Humana Press
- Additional Links
- Industry Sectors
- Editor Affiliations
- 1. Dental and Medical Schools of Guy’s, King’s, and St. Thomas’s Hospitals, King’s College
- Author Affiliations
- 2. School of Biological Sciences, University of Manchester, UK
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